1. Field of the Invention
This invention relates to agents and therapy to lessen morbidity and mortality by protecting against septic shock and Adult Respiratory Distress Syndrome (ARDS). More specifically, the invention relates to therapy with Protein Kinase C (PKC) inhibitors to protect against septic shock and reduce ARDS.
2. Description of the Prior Art
Septic shock is defined in the medical dictionaries as a type of shock associated with overwhelming infection. Most commonly, the infection is produced by gram-negative bacteria although other bacteria, viruses, fungi and protozoa may also be causes. As summarized in Infectious Diseases and Medical Microbiology, 2nd edition, edited by Braude et al., Chapter 92, pages 700 et seq.
"Shock is a syndrome of generalized metabolic failure resulting from prolonged inadequacy of tissue perfusion. Its early clinical manifestations reflect malfunction of those organs most dependent on uninterrupted blood flow, particularly the brain, as well as compensatory adjustments designed to maintain adequate arterial pressure. As these adjustments fail, urinary output decreases and biochemical indices of distorted metabolism are detectable; specifically non-oxidative glycolysis with low yield of high energy chemical bonds testifies to the widespread nature of the disorder. In the end, it is the failure of energy production rather than damage to a particular organ that leads to death. PA0 Other terms, such as `circulatory collapse,` `circulatory failure,` and `hypoperfusion,` have been substituted for `shock` in an attempt to pinpoint the specific nature of the derangement. When it occurs as a specific complication of infection, it is referred to as `infectious shock,` `septic shock,` `bacteremic shock,` and even `endotoxin shock.` The last three terms specifically implicate bacterial infection and are therefore too restrictive. Because `infectious shock` is sufficiently broad as well as concise, this term will be used in the present chapter. PA0 Shock may occur in the course of almost any severe infection, but it is particularly characteristic of bacteremia due to gram-negative bacilli. . . . The importance of endotoxin, the lipopolysaccharide (LPS) composing part of all gram-negative cell walls, is readily apparent because it produces a similar syndrome in experimental animals. Partly because of the extensive use of endotoxin as an investigative tool, endotoxin shock is commonly regarded as the prototype of infectious shock."
The shock is believed to be caused by the action of endotoxins, other products of the infectious agent, or host mediators released in response to the infectious agent on the vascular system. Such action causes altered patterns of perfusion of tissues, and large volumes of blood to be sequestered in the capillaries and veins.
Research by others into PKC inhibition and treatment of inflammatory responses have disclosed that endothelial cells express adhesive proteins in response to sepsis associated stimuli such as endotoxin and cytokines such as interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF). Magnuson et al. (1) and Lane et al. (2) have shown that these adhesive proteins can be reduced on endothelial cell surfaces by inhibition of PKC with staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7). Surface presentation of these adhesive proteins enhances white blood cell infiltration and activation which can result in tissue damage in high inflammatory states like septic shock. In addition, PKC activation enhances endothelial cell permeability resulting in edema. This response to inflammatory agents was also abrogated by exposure of the cells to the PKC inhibitor H7 (3).
Both endotoxin and endotoxin induced mediators (e.g. IL-1 and TNF) have significant effects on vascular smooth muscle contractile performance (13,14). These agents reduce blood vessel contractile strength and sensitivity to agonists that regulate blood pressure; therefore contributing to the poor tissue perfusion characteristic of septic shock. The use of PKC inhibitors to protect vascular smooth muscle against endotoxin and mediator effects on contractile function has not been demonstrated.
Merrill et al. (19) identified that the lipid, sphingosine, is capable of inhibiting PKC in vivo and in vitro. Sphingosine analogues have also been reported to have PKC inhibitory activity (42). Hannun et al. (43) discuss the functions of sphingolipids and sphingolipid breakdown products. The sphingolipids' properties as a PKC inhibitor are discussed as well as the possible pharmacological applications of that inhibition potential as tools to study PKC and its functions. Sphingosine has significant effects on cell physiology (19,43) and is metabolizable because it is a natural membrane component of cells. The usefulness of sphingosine and its analogues as therapies in vivo has not been demonstrated especially in relationship to septic shock and vascular regulation.
Investigations into the influence of PKC in vascular smooth muscle function have primarily been concerned with delineating the role of PKC in contraction. Protein kinase C modulates many aspects of receptor and non-receptor mediated contractile signal transduction and produces diverse effects on vascular cell contractile responsiveness and calcium homeostasis. Activation of PKC with phorbol esters results in enhanced sensitivity of the contractile mechanism to Ca.sup.2+ and therefore augments contraction in ferret and rat aortas and the rat mesenteric artery (4,5). Activation of PKC by phorbol diesters increases cellular Ca.sup.2+ influx in rat and rabbit aortas (6,7). In contrast, in cultured rat aortic smooth muscle cells, phorbol esters enhance Ca.sup.2+ efflux via a plasma membrane Ca.sup.2+ pump (8), and inhibit influx through voltage-dependent Ca.sup.2+ channels (9). Phorbol ester treatment of rabbit aortic smooth muscle cells enhances Ca.sup.2+ efflux, abolishes the Ca.sup.2+ efflux that normally occurs in response to norepinephrine, and significantly reduces .alpha..sub.1 -adrenergic receptor number (10). In cultured rat aortic smooth muscle cells, exposure to a phorbol ester increases Ca.sup.2+ efflux from the cells and inhibits angiotensin II induced breakdown of PIP.sub.2 without altering receptor number or affinity (11). Treatment of rat aortic smooth muscle cells with a phorbol ester attenuate PIP.sub.2 hydrolysis and Ca.sup.2+ efflux in response to vasopressin; in this study the phorbol ester also had no effect on receptor number and it is suggested that the diminished responsiveness possibly results from the uncoupling of vasopressin receptors from guanine nucleotide binding protein (12).
Treatment of septic shock is complex, requiring therapies directed at ameliorating the source of infection [antibiotics], blocking effects of products of the infectious agent and inflammatory mediators on tissues [anti-endotoxin (Young et al. U.S. Pat. No. 4,918,163) and anti-cytokine agents [(Mandell et al. U.S. Pat. No. 4,965,271)], and maintenance of cardiovascular function [volume expansion and pressor agents]. However, mortality still runs at about 100,000 patients per year (40 to 50% of those in shock) and no therapies are available to prevent vascular contractile defects.